Production of chemicals through cultivation of microorganisms is generally performed under well-controlled conditions wherein a constant environment regarding temperature, oxygen concentration and pH are maintained. In addition, culture vessels, which include the microorganisms, are fed with different types of nutrients that cells use for growth and the production of desired chemical compounds. Under these controlled conditions, certain metabolic pathways and physiological responses programmed into a cell's genome are unnecessary, and in fact, may interfere with the optimized performance of a preferred process. In bacterial cells a number of reactions in these metabolic pathways and physiological responses can be eliminated or enhanced to improve the production of desired products.
FIG. 1 illustrates a number of pathways and reactions involved in the assimilation of glucose. Enhanced efficient glucose assimilation can result in increased energy production, such as ATP, NADH, NADPH and FADH, for use by a bacterial strain. The increased energy production can then result in an increase in production and/or growth per unit weight of generating biomass or per unit weight of provided carbon substrate.
Generally, increased glucose assimilation and metabolite production in microorganisms by genetic engineering has involved inactivation or deregulation of specific enzymes; overexpression of genes that are involved in central metabolic routes, such as glycolysis and the TCA cycle; the introduction of heterologous genes; and various other approaches. However, microbial strains regulate their physiology not just by controlling specific steps in a pathway, but also by coordinating a number of biosynthetic and catabolic pathways through the use of global regulators. One of these global regulatory systems is the ArcB/ArcA regulatory system. Another global regulatory system is the RpoS system.
In particular, in facultative anaerobic bacteria, such as E. coli, some of the enzymes of the tricarboxylic acid (TCA) cycle are repressed under anaerobic conditions and particularly when the microorganisms are grown on glucose. The response of the TCA cycle enzymes in the absence to oxygen is regulated mainly at the transcriptional level by the ArcB/ArcA two-component regulatory system (NCBI entries NP 418818 (AE000400) and NP 417677 (AE000510)). ArcB (histidine kinase) senses the absence of oxygen and subsequently phosphorylates the regulator ArcA. The phosphorylated-ArcA then binds to regulatory regions, which are operably linked to the coding regions of TCA cycle regulated enzymes and either activates or represses transcription. Examples of enzymes regulated in this manner include succinate dehydrogenase, 2-oxoglutarate dehydrogenase, isocitrate dehydrogenase, and citrate synthase, which are all repressed by phosphorylated ArcA. (Lynch et al., (1996) in REGULATION OF GENE EXPRESSION IN ESCHERICHIA COLI. Eds. Lin et al., Chapman and Hall, New York). In arcA mutants, TCA cycle genes that were repressed by arcA become derepressed. (luchi et al., (1988) PNAS USA 85:1888-1892). Prohl et al. compared the production of products and specific enzyme activities in an E. coli wild-type strain and an E. coli arcA mutant strain, wherein the arcA was inactivated when the strains were grown on glucose or glycerol in the presence of oxygen or nitrate. (Prohl et al., (1998) Arch. Microbiol. 170:1-7). The authors concluded that the strong repression of the TCA cycle during nitrate respiration (anaerobic conditions) occurs only during growth on glucose but not on other substrates, such as glycerol. However, deletion of arcA did not have any affect on E. coli strains when grown under aerobic conditions.
RpoS genes encode DNA-dependent RNA polymerase sigma subunits and when bound to the core RNA polymerase, allows the transcription of a subset of genes. For example in E. coli, rpoS is induced under various stress conditions and controls the expression of a catalase gene (katE) and glycogen biosynthesis. RpoS is believed to be a global regulator of the general stress response in bacteria and can be triggered by many different stress signals. Often, activation of rpoS is accompanied by a reduction or cessation of bacterial growth. This mechanism provides the host microorganism with the ability to survive the encountered stress as well as potential other stresses. (Hengge-Aronis, R. (2002) Microbiol. Mol. Biol. Rev. 66:373-395).
The major function of the general stress response is preventive. There are more than 70 rpoS dependent genes known and the majority of these confer resistance against oxidative stress, near-UV radiation, potentially lethal heat shock, hyperosmolarity, acidic pH and organic solvents. Compared with wild-type rpoS strains, rpoS mutants were unable able to induce the general stress response in bacteria and could not survive prolonged starvation periods (Hengge-Aronis R. (2002) Microbiol. Mol. Biol. Rev. 66:373-395). In recombinant bacterial strains, the production of protein during culture, and particularly during late stage fed-batch fermentation, may result in a general stressful condition, which may induce the RpoS-dependent response. It has been demonstrated that in continuous culture, at low dilution rates of, for example 0.13 h−1, a rpoS− strain produced more recombinant protein than the wild-type strain. However, the effect was promoter dependent, and at higher dilution rates (0.38 h−1) the mutation had no effect on the production of recombinant protein. (Chou et al., (1996) Biotechnol. Bioeng. 50:636-642). It is believed the effect of a rpoS mutation on the production of metabolites has not been reported.
The phosphoenolpyruvate carboxylase (Ppc) enzyme (E.C. 4.1.1.31/32/38/49) catalyzes the conversion of phosphoenolpyruvate (PEP) to oxaloacetate (OAA). Ppc replenishes the TCA cycle by directly providing OAA, which may have been removed for other biosynthetic reactions. If OAA is not replenished, the efficiency of the TCA cycle may be diminished. E. Coli cells having an inactivated ppc (ppc−) grew slower than wild-type ppc− cells (ppc+), and the ppc+ cells used glucose about three times as efficiently as the ppc− cells. However, despite this effect, the ppc− cells overproduced phenylalanine (Phe). The ppc− cells produced at least 16 times more Phe than the ppc+ cells. The production of unwanted by-products, such as acetate and pyruvate were also stimulated in the ppc− cells. (Miller et al., (1987) Microbiol. 2:143-149). In another study the overproduction of Ppc in E. coli was effected by cloning the ppc in a multicopy plasmid under a tac promoter resulting in an increase in growth yield on glucose as well a decrease in the secretion of organic acids. (Liao et al., (1993) Appl. Environ. Microbiol. 59:4261-4265). The above results suggest that the wild-type level of Ppc is not optimal for the most efficient glucose utilization in batch cultures. Overexpression of Ppc is expected to favor the production of amino acids of the OAA family, such as Asp, Met, Lys, Thr and Ile.
By modification of a global regulator such as arcA or rpoS and optionally by overexpression of ppc, a more efficient metabolic pathway for glucose assimilation may be achieved in a bacterial microorganism.